Study On The Mechanism Of Quinclorac Resistance In Echinochloa Crus-galli Var.zelayensis

Echinochloa crus-galli var.zelayensis,a variant of E.crus-galli(L.)Beauv.,is seriously harmful to rice production in the middle and lower reaches of the Yangtze River in China.Quinclorac is a selective,auxin-type,and effective herbicide that has been used in China to control E.spp.for about 30 years.Auxin-type herbicides,including quinclorac,can mimic the plant endogenous auxin,IAA,first by binding to the receptor TIR1/AFB and the co-receptor Aux/IAA,and then inducing the accumulation of downstream substances,including ethylene,cyanide and abscisic acid(ABA),each of which can cause the abnormal growth of plants.The previous studies in of our laboratory have shown that the E.crus-galli var.zelayensis biotypes,JCWJ-R1,selected from the paddy field of Wujin District,Changzhou City,Jiangsu Province and SSXB-R2,selected from paddy fields of Songjiang District,Shanghai City,have evolved resistance to quinclorac.The RI(resistance index)of the two resistant biotypes reached 66.9 and 9.7,respectively,compared to the sensitive biotype collected from Jiangsu Academy of Agricultural Sciences(Nanjing City,Jiangsu Province),JNNX-S.It was preliminarily clarified that the mechanism of quinclorac resistance in E.crus-galli var.zelayensis was closely related to the ethylene biosynthesis at the physiological and biochemical level.However,the molecular mechanism of the inhibition of ethylene biosynthesis in quinclorac-resistant weeds including E.crus-galli var.zelayensis,remains unclear.The molecular mechanism of detoxification of cyanide in quinclorac-resistant plants has not been reported.In addition,the relationship between the biosynthesis of ABA in grass weeds and quinclorac resistance also need to be studied.The present study takes the two quinclorac-resistant E.crus-galli var.zelayensis biotypes,JCWJ-R1 and SSXB-R2,as research materials and takes the sensitive biotype,JNNX-S,as the control to study the mechanism of resistance.The resistance level of two resistant biotypes to quinclorac and other auxin-type herbicides were studied and then the differences in genes encoded auxin receptor,TIR1 and co-receptor,IAA16 between quinclorac-resistant and-sensitive biotypes was explored;using an ethylene biosynthesis inhibitor,the molecular mechanism of the relationship between ethylene biosynthesis and quinclorac resistance was studied;the relationship between the molecular mechanism of the enhanced ability of cyanide metabolism was researched;by determining the photosynthesis and oxidative stress related indicators,the relationship between the biosynthesis of ABA and quinclorac resistance was also studied.The main results are as follows.1.The whole-plant pot bioassay was used to detect the resistance level of the quinclorac-resistant biotypes to quinclorac and other common auxin-type herbicides.It indicates that JCWJ-R1 and SSXB-R2,were still resistant to quinclorac after seed reproduction and the RI of the above two biotypes were 12.5 and 139.7 compared to the sensitive biotypes,which has reached the moderately resistance level and highly resistance level,respectively.The two resistant biotypes were also resistant to quinmerac and quintrione and the RI were 33.0 and 95.7,26.2 and 36.6,respectively.There were almost no differences in the sensitivity of the three biotypes to other auxin-type herbicides.Our results indicated that the resistance level can be inherited in the two resistant biotypes,and the two resistant biotypes evolved cross-resistance to quinmerac and quintrione but no resistance to other auxin-type herbicides were found.2.The results of studies on the relationship between differences in the auxin receptor gene,EcTIRl,and the auxin co-receptor gene,EcIAA16,and the resistance mechanism showed that:there was no difference in the gene sequence and mRNA levels of EcTIR1 in JCWJ-R1 and SSXB-R2,compared to JNNX-S;the mRNA level of EcIAA16 which is reported to closely related to auxin-type herbicides resistance was significant higher in JCWJ-R1 and SSXB-R2,which were 4.9-times and 4.7-times compared to JNNX-S,respectively;the increase in transcript level of EcIAA16 was related to the resistance level after quinclorac treatment,reaching to 10.4 times and 3.3 times compared to respective control in JNNX-S and JCWJ-R1,and no increase in SSXB-R2;there was no mutations in EcIAA16 sequence of JCWJ-R1 and SSXB-R2 compared to JNNX-S.It indicated that the quinclorac resistance was not caused by the structural changes of EcTIRl and EcIAA16,but the differences in mRNA levels of EcIAA16 between resistant and sensitive biotypes may be involved in the mechanism of quinclorac resistance.3.The results of studies on the mechanism of resistance induced by the inhibition of ethylene biosynthesis and its molecular mechanism are shown as follow:The quinclorac and ethylene synthesis inhibitor had no effect on the fresh weight of SSXB-R2,but quinclorac treatment caused a significant decrease in the fresh weight of the JCWJ-R1 and JNNX-S.However,the fresh weight of JCWJ-R1 pretreated with the ethylene synthesis inhibitor did not decrease any more after quinclorac treatment,and the extent of the decline in fresh weight of JNNX-S pretreated with the ethylene synthesis inhibitor decreased after quinclorac treatment.Quinclorac treatment had no effect on the releasing of ethylene in SSXB-R2,but caused a significant increase in the releasing of ethylene in JCWJ-R1 and JNNX-S,especially in JNNX-S whose maximum releasing of ethylene reached 5.3 times compared to the control.JCWJ-R1 and SSXB-R2 pretreated with ethylene inhibitor had no increase in ethylene releasing after quinclorac treatment,and the ethylene releasing were significantly lower than those with quinclorac treatment only at any corresponding treatment time-points.However,the amount of ethylene releasing in JNNX-S pretreated with the ethylene synthesis inhibitor still increased after quinclorac treatment,but the maximum amount of ethylene releasing was only 40%of that treated with quinclorac only.The expression levels of 3 EcACSs and 7 EcACOs genes which are the key genes in ethylene biosynthesis obtained from E.crus-galli var.zelayensis were not induced by quinclorac in SSXB-R2 and also showed no difference in the expression trends compared to other two biotypes.However,the expression levels of EcACS7 and EcACO1 were induced by quinclorac to 47.7 times and 34.1 times compared the control in JCWJ-R1,and expression levels of EcACS-like,EcACS7 and EcACO1 were increased by 26.1,556.3 and 47.2 times in JNNX-S,after quinclorac treatment.Pretreatment with the ethylene synthesis inhibitor could significantly reduce the increase in the expression of EcACS-like and EcACS7 in JCWJ-R1 and JNNX-S.It was further verified that the mechanism of quinclorac resistance in E.crus-galli var.zelayensis is closely related to the ethylene biosynthesis.It was found that the different expression patterns of EcACS-like,EcACS7 and EcACO1 among three biotypes led to differences in ethylene production.The lower the level of gene expression led to the lower the amount of ethylene releasing,resulting in the higher level of resistance to quinclorac,which may be an important mechanism of quinclorac resistance.4.The results of studies on the relationship between the enhanced ability of cyanide metabolism and mechanism of quinclorac resistance,and its molecular mechanism showed that:the activity of the ?-cyanoalanine synthesis(?-CAS)which can degrade cyanide was significantly higher in JCWJ-R1 and SSXB-R2 than that in JNNX-S;one single-nucleotide difference at 884 position was detected in the EcCAS encoding ?-CAS of JCWJ-R1 and SSXB-R2 compared with JNNX-S,which caused an amino acid substitution,replacing Met with Lys at 295 position in JCWJ-R1 and SSXB-R2;the mRNA level of EcCAS was higher in JCWJ-R1 and SSXB-R2 than that in JNNX-S.The mRNA level of ADP/ATP carrier protein(ANT)gene that was closely related to energy balance,could be strongly induced by quinclorac in JNNX-S,while no significant changes in JCWJ-R1 and SSXB-R2 were detected.It was speculated that ?-CAS may play a crucial role in the mechanism of quinclorac resistance in E.crus-galli var.zelayensis.The mutation and high mRNA level of EcCAS in JCWJ-R1 and SSXB-R2 may be the molecular basis of higher ?-CAS activity,so resistant biotypes with stronger cyanide metabolism ability led to avoid the damage of cyanide to energy balance,and then reduce or avoid the damage caused by quinclorac.5.The results of studies on the relationship between mechanism of ABA biosynthesis and mechanism of quinclorac resistance shows that:quinclorac treatment induced less ABA production and less change rate of ABA production in JCWJ-R1 and SSXB-R2 compared with JNNX-S,and after 24 h of quinclorac treatment,the ABA content in JNNX-S was 46.1 times compared to the control,and 1.3 times and 3.1 times compared to the maximum of ABA content in JCWJ-R1 and SSXB-R2;the 9-cis-epoxycarotenoid dioxygenase(NCED)gene,which is closely related to ABA biosynthesis,could be induced by quinclorac in all three biotypes,and there was no difference in expression patterns among the three biotypes;quinclorac treatment led to a rapid and significant decline in the content of chlorophyll a/b and dry weight in JNNX-S but no any effect on SSXB-R2;transcriptome sequencing of JNNX-S and SSXB-R2 showed that more differentially expressed genes appeared in JNNX-S after quinclorac treatment,and the ratio of the number of quinclorac-regulated genes to the total number of genes in the pathway was the largest in three pathways related to photosynthesis,and then RT-qPCR verified the transcript levels of 34 photosynthesis-related candidate genes,the mRNA levels of which were induced decline by quinclorac in JNNX-S,less affected or rapid recovery after decline in SSXB-R2;after quinclorac treatment,the malondialdehyde content in JNNX-S was 2.8 times of the control,which was 2.1 and 2.2 times of that in JCWJ-R1 and SSXB-R2,but the number of antioxidase genes induced to increase by quinclorac in JCWJ-R1 and SSXB-R2 were less than JNNX-S.It was speculated that less ABA caused less interference in photosynthesis and lower oxidative stress,which may be also involved in quinclorac resistance,but the increase in antioxidant capacity may be the common response after oxidative stress and not the mechanism of quinclorac resistance in E.crus-galli var.zelayensis.In conclusion,the present study indicated that the differences in transcript level of auxin co-receptor gene ECIAA16 between resistant and sensitive biotypes may affect the binding of quinclorac to resistant E.crus-galli var.zelayensis,which may make the downstream EcACS-like,EcACS7,and EcACO1 genes not to be induced to transcribe and then the biosynthesis of ethylene to be inhibited,and lead to the inhibition of ABA biosynthesis that is another response pathway,avoiding photosynthesis inhibition and oxidative stress,also involved in the quinclorac resistance.In addition,due to the mutation in EcCAS or higher mRNA levels,the resistant E.crus-galli var.zelayensis may had a stronger ability to metabolize cyanide,which was also beneficial to avoid harmful effects and avoid more ethylene production.